A New Single Particle Electron Microscopy Reconstruction Methodology is developed by LBNL Researchers


Researchers led by Professor Eva Nogales at LBNL and UC-Berkeley have developed a methodology to improve the quality of 3-D reconstructions of macromolecular complexes produced by electron microscopy and image analysis. Many of these complexes are the molecular machines that perform vital biological functions in the cell; accurately visualizing them provides key insights into how they operate. Generating reliable initial molecular models is a critical step in the reconstruction of asymmetric complexes by 3D electron microscopy. This is made particularly difficult because many macromolecules are intrinsically flexible and exist in multiple conformations in solution. The most robust standard method, Random Conical Tilt (RCT), uses geometrical principles to generate different views of the object under study and to put those images together into a 3D structure. However, it has the serious limitation of producing reconstructions that are distorted due to the absence of certain views of the complex. Substantial effort and expertise is required to minimize the impact of this missing cone of data. The Nogales Laboratory has developed a novel approach, termed the Orthogonal Tilt Reconstruction method (OTR) that eliminates the missing cone altogether by collecting data at +45° and -45° tilts between sample and microscope. One tilted data set is used for alignment and classification and the other set which provides views orthogonal to those in the first is used for reconstruction. The absence of a missing cone in OTR reconstructions makes it more straightforward to detect and characterize conformational flexibility in macromolecular complexes.